1. Valyl-tRNA Synthetase Josh Jarodsky http://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/images/aaRS.gif

2. General Information Valyl-tRNA Synthetase = ValRS ValRS is an enzyme aminoacyl-tRNA synthetase E.C. 6.1.1.9 PDB: 1IVS Seen in all Domains of Life: Archaea – Haloferax volcanii Bacteria - Escherichia Coli Eukaryote - Homo Sapiens, Saccharomyces cerevisiae Important for translation of mRNA for protein synthesis PDB: 1IVS Valine-tRNA Ligase, BRENDA Enzyme Database, http://www.brenda-enzymes.org/enzyme.php?ecno=6.1.1.9

3. Reaction of ValRS Binds L-Valine & tRNA(Val) to form Valyl-tRNA ATP cofactor http://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpghttp://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpgModified version of http://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.htmlhttp://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.html

4. Structure: PDB ID: 1IVS ValRS+AMS+tRNA(Val) Asymmetric Homodimer o 2 Subunits o 4 Domains per sub unit Large crescent shaped binding pocket 37 Alpha helices; 380 residues 42 Beta strands; 129 residues PDB: 1IVS Asymmetric Mirror Plane Secondary Structure:

5. Structure: ValRS+tRNA(Val)+ATP+L-Valine PDB: 1IVS L-Valine+AMS ValRS tRNA(Val)

6. Primary Structure and Amino Acid Sequence Alignment Comparison of 10 Organisms Large amounts of conservation: ATP binding site L-Valine binding site tRNA(Val) anticodon binding sites tRNA(Val) binding sites Plus others not focused on http://www.brenda-enzymes.org/sequences.php?f[stype_ec]=1&f[ec]=6.1.1.9

7. Interesting Conserved Amino Acids KMSKS Loop Lysine, Methionine, Serine, Lysine, Serine Characteristic amino acid sequence for the catalytic site of aminoacyl-tRNA synthetases for ATP and AA binding Hountondji, C. et al. (2002) Crucial Role of Conserved Lysine 277 in the Fidelity of tRNA Aminoaclation by Escherichia coli Valyl-tRNA Synthetase. BioChem, 41, 14856-14865.

8. KMSKS Loop PDB: 1IVS K528-S532 L-Valine + AMS (Yellow, AMS is an ATP analog) tRNA (Orange) Ser530 Lys531 Lys528 Met529 Ser532

9. Binding Site: ValRS+tRNA(Val) PDB: 1IVS tRNA(Val)

10. Binding Site: ValRS+tRNA(Val) PDB: 1IVS Arg570 – tRNA Backbone interaction CCA Terminal Binding Anti-Codon Interactions Fukai, S. et al. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803.

11. CCA Terminal Interactions: C974(Grey) –side chain interactions with: Glu261,281 (Magenta) Leu278 (Red) C975(Grey) –”Contacts with:” Glu261 (Magenta) Phe264 (Blue) A976(Grey)- H-Bond with: Tyr337 (Green) A976 (Grey)-Sandwiched between: Leu269 (Red) Phe264 (Blue) Binding Site: 3’ End of tRNA (5’-CCA-3’) PDB: 1IVS Fukai, S. et al. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803. Glu281 Leu278 Leu269 Phe264 Glu261 Tyr337

12. Anticodon Interactions Asn584 o H-Bonds with hydroxyl groups on: C933 A934 o Side Chain interactions with: Phe588 (Blue) Leu650 (Red) Cys646 (Yellow) PDB: 1IVS Anti-Codon: CAC Minor Groove Interactions Fukai, S. et al. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803. Lys581 Asn584 Phe588 Cys646 Leu650 C935 o Hydrogen Bonding N3 to Lys581 Phe588

13. Interesting Conserved Amino Acids Lysine270 Involved in editing Edits by nucleophilic attack Second part of the “Double Sieve” Hountondji, C. et al. (2002) Crucial Role of Conserved Lysine 277 in the Fidelity of tRNA Aminoaclation by Escherichia coli Valyl-tRNA Synthetase. BioChem, 41, 14856-14865.

14. Lysine270 tRNA Interaction PDB: 1IVS Lys270

15. Lysine270 tRNA Interaction Lys270 (Yellow) Up and Down stream ValRS AA (Green) A975 (Red) Down stream tRNA Nucleic Acids (Orange) Interaction: o Amine group of Lys270 would preform a nucleophilic attack on improper amino acid PDB: 1IVS Lys270 A975 Hountondji, C. et al. (2002) Crucial Role of Conserved Lysine 277 in the Fidelity of tRNA Aminoaclation by Escherichia coli Valyl-tRNA Synthetase. BioChem, 41, 14856-14865

16. Double Sieve Fukai, S. et al. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L- Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803. Valine Isoleucine Threonine

17. ValRS Proofreading and Editing Pathways Gruic-Sovulj, I. et al. (2007) Hydrolysis of non-cognate aminoacyl-adenylates by a class II aminoacyl- tRNA synthetase lacking an editing domain. FEBS Letters, 581, 26, 5110-5114

18. Why should we care? Without ValRS: Valine would not be added to any protein sequence This would change proteins sequence leading to: Non-native conformations Malfunctioning protein Nonfunctional protein Malfunctioning ValRS: This would change proteins sequence leading to: Non-native conformations Malfunctioning protein Nonfunctional protein All of which could result in the death of the affected organism

19. Conclusions Valyl-tRNA Synthetase is an important enzyme o Seen in all domains of life o Large sections of conserved amino acids Structure o Asymmetric homodimer o Large crescent shape o Mainly alpha helices Binding o L-Valine + ATP and catalysis via KMSKS Loop o tRNA Backbone Minor groove CCA terminal Anti-codon Editing o Multiple pathways of editing pre and post transfer of tRNA o Lysine270 Why is it Important? o Accurate translation of mRNA for correct protein sequence o Proper conformations and functionality

20. Work Cited 1)http://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/ima ges/aaRS.gifhttp://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/ima ges/aaRS.gif 2)PDB 1IVS 3)http://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpghttp://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpg 4)http://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.htmlhttp://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.html 5)http://www.brenda-enzymes.org/sequences.php?f[stype_ec]=1&f[ec]=6.1.1.9http://www.brenda-enzymes.org/sequences.php?f[stype_ec]=1&f[ec]=6.1.1.9 6)Hountondji, C.; Lazennec, C.; Beauvallet, C.; Dessen, P.; Pernollet, J.; Plateau, P.; Blanquet, S. (2002) Crucial Role of Conserved Lysine 277 in the Fidelity of tRNA Aminoaclation by Escherichia coli Valyl-tRNA Synthetase. BioChem, 41, 14856-14865. 7)Fukai, S.; Nureki, O.; Sekine, S.; Shimada, A.; Tao, J.; Vassylyev, D.; Yokoyama, S. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L- Isoleucine and L-Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803. 8)Gruic-Sovulj, I., Rokov-Plavec, J., Weygand-Durasevic, I. (2007) Hydrolysis of non-cognate aminoacyl-adenylates by a class II aminoacyl-tRNA synthetase lacking an editing domain. FEBS Letters, 581, 26, 5110-5114